Violet laser excitable dyes and their method of use

ABSTRACT

The present invention provides dye compounds optimally excited at about 400 nm and have a Stokes shift of at least about 80 nm. These dyes find use in detection of analyte in a sample and the preparation of dye-conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/444,023filed Jul. 28, 2014, which is a divisional of U.S. application Ser. No.13/777,910 filed Feb. 26, 2013, (now U.S. Pat. No. 8,822,695 issued Sep.2, 2014), which is a continuation of U.S. application Ser. No.13/149,392 filed May 31, 2011 (now U.S. Pat. No. 8,415,477 issued Apr.9, 2013), which is a continuation of U.S. application Ser. No.11/535,458 filed Sep. 26, 2006 (now U.S. Pat. No. 8,158,801 issued Apr.17, 2012), which claims priority to U.S. Provisional Application Ser.No. 60/745,599 filed Apr. 25, 2006 and U.S. Provisional Application Ser.No. 60/720,690, filed Sep. 26, 2005; which disclosures are hereinincorporated by reference.

INTRODUCTION

Field of the Invention

The invention relates to colored and fluorescent dyes, includingreactive dye derivatives, and dye-conjugates; and to their use instaining samples and detecting ligands or other analytes. The inventionhas applications in the fields of cell biology, neurology, immunology,pathology and proteomics.

Background of the Invention

Fluorescent dyes are widely used as tracers for localization ofbiological structures by fluorescence microscopy, for quantification ofanalytes by fluorescence immunoassay, for flow cytometric analysis ofcells, for measurement of physiological state of cells and otherapplications (Kanaoka, Angew. Chem. Intl. Ed. Engl. 16: 137 (1977);Hemmila, Clin. Chem. 31: 359 (1985)). Among the advantages offluorescent agent over other types of absorption dyes include thedetectability of emission at a wavelength distinct from the excitation,the orders of magnitude greater detectability of fluorescence emissionover light absorption, the generally low level of fluorescencebackground in most biological samples and the measurable intrinsicspectral properties of fluorescence polarization (Jolley et al., Clin.Chem. 27: 1190 (1981)), lifetime (U.S. Pat. No. 4,374,120) and excitedstate energy transfer (U.S. Pat. Nos. 3,996,345; and 4,542,104).

Fluorescent agents are now widely used to determine physiologicalfunctions in patients during routine checkups or diagnostic procedures,to monitor the exposure of workers and others to potentially harmfulchemicals such as toxic or carcinogenic pesticides or inorganicmaterials in the atmosphere, soil, or drinking water, in determining theeffectiveness of pharmaceuticals on disease states or conditions, inscreening new compounds for biological activity as either promoters orinhibitors of particular enzymes, in monitoring gene and transgeneexpression, and in performing immunological and other laboratory assayssuch as enzyme-linked immunosorbent assays (ELISAs) and Western blots.

Optical methods of detection, such as fluorescence emission, UVabsorptivity, and colorimetry are convenient and highly effective fordetecting, monitoring, and measuring fluorescent agents, since methodssuch as these can generate either qualitative or quantitativeinformation and detection can be achieved either by direct visualobservation or by instrumentation.

For many applications that utilize fluorescent dyes as tracers, it isnecessary to chemically react the dye with a biologically active ligandsuch as a cell, tissue, protein, antibody, enzyme, drug, hormone,nucleotide, nucleic acid, polysaccharide, lipid or other biomolecule tomake a fluorescent ligand analog or to react the dye with natural orsynthetic polymers. With these synthetic probes, the biomoleculefrequently confers a specificity for a biochemical interaction that isunder investigation and the fluorescent dye provides the method fordetection and/or quantification of the interaction. Thus, useful dyesare based on a versatile fluorescent nucleus that allows the preparationof reactive derivatives of several different types that exhibitreactivity toward a variety of chemically reactive sites.

There is a recognized need for suitable fluorophores, particularlyreactive fluorophores, for applications in multi-color, multiplexedapplications, such as microscopy, flow cytometry, immunoassays, andnucleic acid sequencing. Presently there exists a need for a dye that isexcited by a violet 405 nm laser, which is not excited by the 488 nmargon laser, with a long Stokes shift allowing for use of multiplelasers.

The existing, violet laser excitable dyes, about 405 nm, generally havea weak absorbtivity (extinction coefficients of less than 20,000 cm⁻¹M⁻¹ at their absorbance maxima), relatively low quantum yields, and/oror not particularly well solubilized in aqueous environments. Suchproperties are less than ideal for a fluorophore of interest forbiological applications. Cascade Yellow is a common violet laserexcitable dye, however this dye is relatively dim when conjugated to acarrier molecule or solid support and has a tendency to aggregate in anaqueous solution.

For example, the wide emission band-widths of many art-recognized dyesresult in significant residual fluorescence background from the violetexcited dyes at wavelengths typically used for detection of fluoresceinemission (typically 515 to 525 nm). Moreover, fluorescence of many ofthe art-recognized dyes is frequently quenched in aqueous solution,resulting in low quantum yields. The lower quantum yield decreases thedetection sensitivity or requires use of disproportionately largerquantities of the less fluorescent dye.

In view of the above, a fluorophore having a reactive group attached tothe fluorescent nucleus of the fluorophore, which is water soluble, andhighly fluorescent within a narrow wavelength range would be a highlydesirable addition to the art-recognized array of reactive fluorophores.The present invention provides such fluorescent agents, conjugatesincorporating the agents and methods of using the agents and theirconjugates.

SUMMARY OF THE INVENTION

Provided are compounds having the structure

X, Y and Z are independently carbon, —CR¹², nitrogen, —NR¹⁴, sulfur,oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S, wherein at least one of X, Y and Z is other thancarbon or C—C.

R¹²-R¹⁵ are independently hydrogen, alkyl, substituted alkyl, alkoxy,hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylicacid, ester, azido, nitro, nitroso, cyano, or thioether.

R¹-R⁷ and R⁹-R¹⁰ are independently is hydrogen, alkyl, substitutedalkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino,aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano,thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-,6- or 7 aromatic or heteroaromatic ring or R¹-R⁷ and R⁹-R¹⁰ comprises areactive group, carrier molecule or solid support. R⁸ is hydrogen,alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁸ comprisesa reactive group, carrier molecule or solid support. Alternatively, amember selected from

-   -   R¹ in combination with R²;    -   R² in combination with R³;    -   R³ in combination with R⁴;    -   R⁴ in combination with R⁵;    -   R⁶ in combination with R⁷;    -   R⁷ in combination with R⁸;    -   R⁸ in combination with R⁹; and    -   R⁹ in combination with R¹⁰;        together with the atoms to which they are joined, form a ring        which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6-        or 7-membered cycloalkyl, a 5-, 6- or 7-membered        heterocycloalkyl, a substituted 5-, 6- or 7-membered        heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-,        6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a        substituted 5-, 6- or 7-membered heteroaryl.

In one embodiment these compounds are excited from about 350 nm to about500 nm, more preferably these compounds are excited with a wavelengthfrom about 380 nm to about 420 nm. Particularly preferred are compounds9A, 9B, 9C and 31.

Also provided are both methods for forming a dye conjugate and methodsfor detecting an analyte in a sample. The method for detecting ananalyte in sample comprises: combining a present compound with a sampleto form a combined sample; incubating the combined sample for asufficient amount of time for the compound to associate with the analytein the sample to form an incubated sample; illuminating the incubatedsample with an appropriate wavelength to form an illuminated sample; anddetecting the illuminated sample whereby the analyte in the sample isdetected.

The method for forming a dye conjugate comprises the steps of: combininga present compound with a carrier molecule or solid support to form acombined sample, wherein the compound comprises a reactive group; andincubating the combined sample for a sufficient amount of time for thecompound to form a covalent bond with either the carrier molecule orsolid support.

Further provided are kits for detecting an analyte in a sample and kitsfor forming a dye conjugate according to the disclosed methods. The kitstypically comprises a present compound and instructions for forming adye conjugate and/or detecting an analyte in a sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the immunophenotyping using Pacific Blue Dye and Compound9.

FIG. 2: Shows the excitation and emission spectra of Compound 9A.

FIG. 3: Shows comparison of Cascade Yellow to Compound 9A with 3different DOL.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H: Show the brighter signal ofCompound 9A mouse anti-human CD8 (FIG. 4A and FIG. 4B) compared toAmCyan mouse anti-human CD8 (FIG. 4C and FIG. 4D) and CD4 (FIG. 4E andFIG. 4F). FIG. 4G and FIG. 4H represent controls.

FIGS. 5A, 5B, 5C and 5D: Show histograms for cells stained with theCompound 31; FIG. 5A) live cells show low fluorescence, FIG. 5B) agedculture cells, FIG. 5C) ethanol-killed cells, and FIG. 5D) heat-killedcells all show higher fluorescence than live cell.

FIGS. 6A, 6B, 6C and 6D: Show dual parameter dot plots demonstratingCompound 31 stain with two calcein AM live cell stains. FIG. 6A) Mixtureof live and heat-killed Jurkat cells stained with calcein green, AM andCompound 31, showing two distinct populations. FIG. 6B) Mixture of liveand ethanol-killed jurkat cells stained with calcein green, AM andCompound 31, showing two distinct populations. FIG. 6C) Mixture of liveand heat-killed Jurkat cells stained with calcein violet, AM andPeriwinkle dead stain, showing two distinct populations. FIG. 6D)Mixture of live and ethanol-killed jurkat cells stained with calceinviolet, AM and Compound 31, showing two distinct populations.

DETAILED DESCRIPTION OF THE INVENTION Introduction

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological analytes in research and diagnostic mixtures. Of particularvalue are methods for measuring small quantities of nucleic acids,peptides (e.g., enzymes), pharmaceuticals, metabolites, microorganismsand other materials of diagnostic value. Examples of such materialsinclude narcotics and poisons, drugs administered for therapeuticpurposes, hormones, pathogenic microorganisms and viruses, antibodies,and enzymes and nucleic acids, particularly those implicated in diseasestates.

The present invention provides dye compounds that have an absorption ofabout 350 nm to about 500 nm and that have a large Stokes shift, makingthem ideal for multiplexing experiments and in certain circumstancesuseful with increasingly popular violet lasers. The compounds are basedon the Cascade Yellow fluorophore. Analogs of the Cascade Yelloe dyeshowed surprising and unexpected results with regard to brightness ofsignal, stability, water solubility and the apparently total loss ofquenching with a high DOS.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of compounds and reference to “a conjugate” includes aplurality of conjugates and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The symbol

, whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), Vogel's Encyclopedia of Practical Organic Chemistry, 5thed., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as, for example, tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

“Su” refers to succinimide, succinimidyl or succinimidyl ester.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom which is amember selected from the group consisting of 0, N, Si, P and S, andwherein the nitrogen, phosphorous and sulfur atoms are optionallyoxidized, and the nitrogen heteroatom is optionally be quaternized. Theheteroatom(s) 0, N, P, S and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂ and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂₋.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms which are members selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “detectable response” as used herein refers to an occurrence ofor a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “dye” as used herein refers to a compound that emits light toproduce an observable detectable signal. “Dye” includes fluorescent andnonfluorescent compounds that include without limitations pigments,fluorophores, chemiluminescent compounds, luminescent compounds andchromophores. The term “fluorophore” as used herein refers to a compoundthat is inherently fluorescent or demonstrates a change in fluorescenceupon binding to a biological compound or metal ion, i.e., fluorogenic.Numerous fluorophores are known to those skilled in the art and include,but are not limited to, coumarin, acridine, furan, indole, quinoline,cyanine, benzofuran, quinazolinone, benzazole, borapolyazaindacene andxanthenes, with the latter including fluoroscein, rhodamine, rhodol,rosamine and derivatives thereof as well as other fluorophores describedin RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS (9^(th) edition, CD-ROM, 2002).

The term “carrier molecule” as used herein refers to a biological or anon-biological component that is covalently bonded to compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-20 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the fluorogenic or fluorescent compounds to anothermoiety such as a chemically reactive group or a biological andnon-biological component. Exemplary linking members include a moietythat includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. Thelinker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

The term “peptide”, as used herein, refers to a polymer in which themonomers are amino acids and are joined together through amide bonds,alternatively referred to as a polypeptide or a protein. When the aminoacids are α-amino acids, either the L-optical isomer or the D-opticalisomer can be used. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Commonlyencountered amino acids that are not gene-encoded may also be used inthe present invention. All of the amino acids used in the presentinvention may be either the D- or L-isomer. The L-isomers are generallypreferred. In addition, other peptidomimetics are also useful in thepresent invention. For a general review, see, Spatola, A. F., inChemistry and Biochemistry of Amino Acids. Peptides and Proteins, B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds., Organic Functional Group Preparations,Academic Press, San Diego, 1989).

The term “salt thereof,” as used herein includes salts of the agents ofthe invention and their conjugates, which are preferably prepared withrelatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The term “sample” as used herein refers to any material that may containan analyte for detection or quantification. The analyte may include areactive group, e.g., a group through which a compound of the inventioncan be conjugated to the analyte. The sample may also include diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like. Typically, the sample is a livecell, a biological fluid that comprises endogenous host cell proteins,nucleic acid polymers, nucleotides, oligonucleotides, peptides andbuffer solutions. The sample may be in an aqueous solution, a viablecell culture or immobilized on a solid or semi solid surface such as apolyacrylamide gel, membrane blot or on a microarray.

The Compounds

In general, for ease of understanding the present invention, the dyecompounds and corresponding substituents will first be described indetail, followed by the many and varied methods in which the compoundsfind uses, which is followed by exemplified methods of use and synthesisof certain novel compounds that are particularly advantageous for usewith the methods of the present invention.

The present compounds are derivatives of the well known fluorophore,Cascade Yellow. Unexpectedly we found that by modifying the corestructure we were able to increase the DOL on dye conjugates withoutsignificant quenching, resulting, in part, in a brighter signal. Inaddition, these compounds demonstrated good water solubility, stabilityand a large Stokes shift.

Cascade Yellow has the following structure:

We found that by modifying the substituent directly attached to thenitrogen, resulted in surprising desirable compounds. Thus, withoutwishing to be bound by a theory, it appears that this modificationdirectly results in the improved characteristics observed with thepresent compounds.

The compounds of the invention have the following structure:

wherein

X is independently carbon, —CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S;

Y is independently carbon, —CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S;

Z is independently carbon, —CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S;

-   -   wherein at least one of X, Y and Z is other than carbon or C—C;    -   R¹² is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,        sulfo, halogen, amino, substituted amino, aldehyde, carboxylic        acid, ester, azido, nitro, nitroso, cyano, or thioether;    -   R¹³ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,        sulfo, halogen, amino, substituted amino, aldehyde, carboxylic        acid, ester, azido, nitro, nitroso, cyano, or thioether;    -   R¹⁴ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,        sulfo, halogen, amino, substituted amino, aldehyde, carboxylic        acid, ester, azido, nitro, nitroso, cyano, or thioether;    -   R¹⁵ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,        sulfo, halogen, amino, substituted amino, aldehyde, carboxylic        acid, ester, azido, nitro, nitroso, cyano, or thioether;

R¹ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R¹ comprises a reactive group, carrier molecule or solid support

R² is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R² comprises a reactive group, carrier molecule or solidsupport;

R³ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R³ comprises a reactive group, carrier molecule or solidsupport;

R⁴ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁴ comprises a reactive group, carrier molecule or solidsupport;

R⁵ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁵ comprises a reactive group, carrier molecule or solidsupport;

R⁶ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁶ comprises a reactive group, carrier molecule or solidsupport;

R⁷ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁷ comprises a reactive group, carrier molecule or solidsupport;

R⁸ is hydrogen, alkyl, substituted alkyl, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁸ comprises a reactive group, carrier molecule or solidsupport;

R⁹ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R⁹ comprises a reactive group, carrier molecule or solidsupport;

R¹⁰ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo,halogen, amino, substituted amino, aldehyde, carboxylic acid, ester,azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic orheteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromaticring or R¹⁰ comprises a reactive group, carrier molecule or solidsupport; or

a member selected from

-   -   R¹ in combination with R²;    -   R² in combination with R³;    -   R³ in combination with R⁴;    -   R⁴ in combination with R⁵;    -   R⁶ in combination with R⁷    -   R⁷ in combination with R⁸;    -   R⁸ in combination with R⁹; and    -   R⁹ in combination with R¹⁰;    -   together with the atoms to which they are joined, form a ring        which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6-        or 7-membered cycloalkyl, a 5-, 6- or 7-membered        heterocycloalkyl, a substituted 5-, 6- or 7-membered        heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-,        6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a        substituted 5-, 6- or 7-membered heteroaryl.

The compounds/embodiments of the invention do not include CascadeYellow:

The middle ring structure of the Cascade Compound is an oxazole ring,however, additional rings will also provide desirable dye compounds, aspresently described. Thus, any 5-, 6- or 7-membered heteroaryl ring,substituted or unsubstituted, is envisioned for this moiety of thecompounds. In particular, X, Y and Z are independently carbon, —CR¹²,nitrogen, —NR¹⁴, sulfur, oxygen, selenium, phosphorous, silicon,arsenic, amido, C—C, CR¹²-CR¹³, NR¹⁴-NR¹⁵, or S—S, wherein at least oneof X, Y and Z is other than carbon or C—C.

R¹²-R¹⁵ are independently hydrogen, alkyl, substituted alkyl, alkoxy,hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylicacid, ester, azido, nitro, nitroso, cyano, or thioether.

R¹-R⁷ and R⁹-R¹⁰ are independently hydrogen, alkyl, substituted alkyl,alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde,carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6-or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromaticor heteroaromatic ring or R¹-R⁷ and R⁹-R¹⁰ independently comprises areactive group, carrier molecule or solid support. R8 is hydrogen,alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R8 comprisesa reactive group, carrier molecule or solid support.

Alternatively, or in addition to any of the above substituents, one ormore of adjacent R¹-R¹⁰ form a fused ring structure. Thus, in one aspecta member selected from R1 in combination with R²; R² in combination withR³; R³ in combination with R⁴; R⁴ in combination with R⁵; R⁶ incombination with R⁷; R⁷ in combination with R⁸; R⁸ in combination withR⁹; and R⁹ in combination with R¹⁰; together with the atoms to whichthey are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl,a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl.

In another more particular embodiment R⁸ is alkyl substituted with —OR′,═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″,—NRSO2R′, —CN and —NO₂; wherein R′, R″, R′″ and R″″ each independentlyare selected from the group consisting of hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted alkyl, alkoxy, thioalkoxy and arylalkyl.

In a more particular embodiment still R⁸ is —(CH₂)₃SO₃ ⁻.

In another embodiment R⁸ has the formula -L-R^(8a), wherein L is absentor a divalent radical selected from the group consisting of alkyl,carbonyl, amino, thio or sulfo; and R^(8a) is selected from the groupconsisting of H, substituted or unsubstituted aryl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted heteroaryl,—SO₃ ⁻, sulfo, sulfonamide, —CN, amino, nitro, halo, hydroxyl, alkoxy,carbonyl, carbonyloxy, aminocarbonyl, carbonylamino, and alkyl; providedR^(8a) is not —CH₂— (C₆H₅)—CONHS.

More particularly, R^(8a) is alkyl or aryl substituted with at least one—SO₃ ⁻. In a more particular embodiment thereof R⁸ is -alkyl-SO₃.

In another embodiment X is —CR¹² (i.e. ═C(R¹²)—), Y is nitrogen, and Zis oxygen. More particularly R¹² is hydrogen.

In another embodiment R⁴ is —SO₃ ⁻.

In another embodiment R³ is a reactive group.

In another embodiment R³ is alkoxy.

Particularly preferred are the compounds having the structure:

wherein, Su is succinimidyl.

Reactive Groups

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support. Thus, inanother aspect of the present invention the compounds comprise thechelating moiety, linker, reporter moiety, a reactive group moiety andoptionally a carrier molecule and/or a solid support.

In an exemplary embodiment, the compounds of the invention furthercomprise a reactive group which is a member selected from an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In a particularembodiment the reactive group is selected from the group consisting ofcarboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide,amine and a maleimide. In exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, and R¹¹, comprises areactive group. Preferably, at least one of R¹, R², R³, R⁴ and R⁵comprises a reactive group. Alternatively, if the present compoundcomprises a carrier molecule or solid support a reactive group may becovalently attached independently to those substituents, allowing forfurther conjugation to a reporter molecule, carrier molecule or solidsupport.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with an aminecontaining molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

These reactive groups are synthesized during the formation of thepresent compound and carrier molecule and solid support containingcompounds to provide chemically reactive dyes. In this way, compoundsincorporating a reactive group can be covalently attached to a widevariety of carrier molecules or solid supports that contain or aremodified to contain functional groups with suitable reactivity,resulting in chemical attachment of the components. In an exemplaryembodiment, the reactive group of the compounds of the invention and thefunctional group of the carrier molecule or solid support compriseelectrophiles and nucleophiles that can generate a covalent linkagebetween them. Alternatively, the reactive group comprises aphotoactivatable group, which becomes chemically reactive only afterillumination with light of an appropriate wavelength. Typically, theconjugation reaction between the reactive group and the carrier moleculeor solid support results in one or more atoms of the reactive groupbeing incorporated into a new linkage attaching the present compound ofthe invention to the carrier molecule or solid support. Selectedexamples of functional groups and linkages are shown in Table 1, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage.

TABLE 1 Examples of some routes to useful covalent linkages ResultingElectrophilic Group Nucleophilic Group Covalent Linkage activatedesters* amines/anilines carboxamides acrylamides thiols thioethers acylazides** amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate estersInorganic azide or alkyl azide phosphine Amide bond *Activated esters,as understood in the art, generally have the formula —COΩ, where Ω is agood leaving group (e.g., succinimidyloxy (—OC₄H₄O₂)sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); oran aryloxy group or aryloxy substituted one or more times by electronwithdrawing substituents such as nitro, fluoro, chloro, cyano, ortrifluoromethyl, or combinations thereof, used to form activated arylesters; or a carboxylic acid activated by a carbodiimide to form ananhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a)and R^(b), which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates.

In some embodiments, the reactive group further comprises a linker, L,in addition to the reactive functional moiety. The linker is used tocovalently attach a reactive functional moiety to the 3-alkoxy xanthenedye/biological moiety compound of the invention. When present, thelinker is a single covalent bond or a series of stable bonds. Thus, thereactive functional moiety may be directly attached (where the linker isa single bond) to the 3-alkoxy xanthene/biological moiety compounds orattached through a series of stable bonds. When the linker is a seriesof stable covalent bonds the linker typically incorporates 1-20nonhydrogen atoms selected from the group consisting of C, N, O, S, andP. In addition, the covalent linkage can incorporate a platinum atom,such as described in U.S. Pat. No. 5,714,327. When the linker is not asingle covalent bond, the linker may be any combination of stablechemical bonds, optionally including, single, double, triple or aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,phosphorus-oxygen bonds, phosphorus-nitrogen bonds, andnitrogen-platinum bonds. In an exemplary embodiment, the linkerincorporates less than 15 nonhydrogen atoms and are composed of acombination of ether, thioether, thiourea, amine, ester, carboxamide,sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.Typically the linker is a single covalent bond or a combination ofsingle carbon-carbon bonds and carboxamide, sulfonamide or thioetherbonds. The following moieties can be found in the linker: ether,thioether, carboxamide, thiourea, sulfonamide, urea, urethane,hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and aminemoieties. Examples of L include substituted or unsubstitutedpolymethylene, arylene, alkylarylene, arylenealkyl, or arylthio.

Any combination of linkers may be used to attach the reactive groups andthe present compounds together, typically a compound of the presentinvention when attached to more than one reactive group will have one ortwo linkers attached that may be the same or different. The linker mayalso be substituted to alter the physical properties of the presentcompounds, such as solubility and spectral properties of the compound.

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or nonbiomolecule) include, but are not limited to, amines,amides, thiols, alcohols, phenols, aldehydes, ketones, phosphates,imidazoles, hydrazines, hydroxylamines, disubstituted amines, halides,epoxides, silyl halides, carboxylate esters, sulfonate esters, purines,pyrimidines, carboxylic acids, olefinic bonds, or a combination of thesegroups. A single type of reactive site may be available on the substance(typical for polysaccharides or silica), or a variety of sites may occur(e.g., amines, thiols, alcohols, phenols), as is typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

Carrier Molecules

In an exemplary embodiment is provided dyes covalently conjugated to acarrier molecule. This includes, but is not limited to, any combinationof compounds disclosed above and any carrier molecule disclosed herein.Typically the compound comprises a reactive group that facilitates thecovalent conjugation to the carrier molecule. The reactive group maycontain both a reactive functional moiety and a linker, or only thereactive functional moiety.

Provided in one embodiment is a first composition that comprises apresent compound, a first fluorophore, and a carrier molecule. Providedin another embodiment is a second composition that includes a firstcomposition in combination with a second conjugate. The second conjugatecomprises a carrier molecule or solid support, as disclosed below, thatis covalently bonded to a second fluorophore. The first and secondfluorophore have different structures and preferably have differentemission spectra. Even more preferably, the first and secondfluorophores are selected so that their fluorescence emissionsessentially do not overlap. In another embodiment the fluorophores havedifferent excitation spectra, alternatively the fluorophores are excitedby the same laser.

The fluorophore on the second conjugate can include substantially anyfluorescent structure known in the art including, but not limited to,small organic fluorophores, fluorescent proteins, and reporter groupsthat are not necessarily fluorescent but which, under correctconditions, convert a fluorogenic substrate into a fluorophore, e.g.,horseradish peroxidase. Exemplary second fluorophores of use in thepresent invention include those that include a moiety that is a memberselected from a coumarin, a xanthene (e.g., fluorescein), a cyanine, apyrene, a borapolyazaindacene, an oxazine, and bimane.

The carrier molecule (or solid support) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various carrier molecules isgenerally applicable to this embodiment of the invention as well asother embodiments.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In another exemplary embodiment, at least onemember selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, and R¹⁰ comprises acarrier molecule or is attached to a carrier molecule. In one aspect atleast one of R¹, R², R³, and R⁴ comprises a carrier molecule or isattached to a carrier molecule.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an IgG binding protein, afluorescent protein, agarose, and a non-biological microparticle.

In some embodiments, the carrier molecule may include a carrier moleculereactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thecarrier molecule reactive functional groups herein.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH²POCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleaved byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is a cell, cellular systems,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and used to the formation of the boundpair. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. In this instance, the dye compounds of the present inventionfunction as a reporter molecule for the specific binding pair. Exemplarybinding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridization

In a particular aspect the carrier molecule is an antibody fragment,such as, but not limited to, anti-Fc, an anti-Fc isotype, anti-J chain,anti-kappa light chain, anti-lambda light chain, or a single-chainfragment variable protein; or a non-antibody peptide or protein, suchas, for example but not limited to, soluble Fc receptor, protein G,protein A, protein L, lectins, or a fragment thereof. In one aspect thecarrier molecule is a Fab fragment specific to the Fc portion of thetarget-binding antibody or to an isotype of the Fc portion of thetarget-binding antibody (U.S. Ser. No. 10/118,204). The monovalent Fabfragments are typically produced from either murine monoclonalantibodies or polyclonal antibodies generated in a variety of animals,for example but not limited to, rabbit or goat. These fragments can begenerated from any isotype such as murine IgM, IgG₁, IgG_(2a), IgG_(2b)or IgG₃.

Alternatively, a non-antibody protein or peptide such as protein G, orother suitable proteins, can be used alone or coupled with albumin.Preferred albumins include human and bovine serum albumins or ovalbumin.Protein A, G and L are defined to include those proteins known to oneskilled in the art or derivatives thereof that comprise at least onebinding domain for IgG, i.e. proteins that have affinity for IgG. Theseproteins can be modified but do not need to be and are conjugated to areactive label in the same manner as the other carrier molecules of theinvention.

In another aspect the carrier molecule is a whole intact antibody.Antibody is a term of the art denoting the soluble substance or moleculesecreted or produced by an animal in response to an antigen, and whichhas the particular property of combining specifically with the antigenthat induced its formation. Antibodies themselves also serve areantigens or immunogens because they are glycoproteins and therefore areused to generate antispecies antibodies. Antibodies, also known asimmunoglobulins, are classified into five distinct c1 asses—IgG, IgA,IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists oftwo identical light polypeptide chains and two identical heavypolypeptide chains (linked together by disulfide bonds).

When IgG is treated with the enzyme papain a monovalent antigen-bindingfragment can be isolated, referred herein to as a Fab fragment. When IgGis treated with pepsin (another proteolytic enzyme), a larger fragmentis produced, F(ab′)₂. This fragment can be split in half by treatingwith a mild reducing buffer that results in the monovalent Fab′fragment. The Fab′ fragment is slightly larger than the Fab and containsone or more free sulfhydryls from the hinge region (which are not foundin the smaller Fab fragment). The term “antibody fragment” is usedherein to define the Fab′, F(ab′)₂ and Fab portions of the antibody. Itis well known in the art to treat antibody molecules with pepsin andpapain in order to produce antibody fragments (Gorevic et al., Methodsof Enzyol., 116:3 (1985)).

The monovalent Fab fragments of the present invention are produced fromeither murine monoclonal antibodies or polyclonal antibodies generatedin a variety of animals that have been immunized with a foreign antibodyor fragment thereof, U.S. Pat. No. 4,196,265 discloses a method ofproducing monoclonal antibodies. Typically, secondary antibodies arederived from a polyclonal antibody that has been produced in a rabbit orgoat but any animal known to one skilled in the art to producepolyclonal antibodies can be used to generate anti-species antibodies.The term “primary antibody” describes an antibody that binds directly tothe antigen as opposed to a “secondary antibody” that binds to a regionof the primary antibody. Monoclonal antibodies are equal, and in somecases, preferred over polyclonal antibodies provided that theligand-binding antibody is compatible with the monoclonal antibodiesthat are typically produced from murine hybridoma cell lines usingmethods well known to one skilled in the art.

In one aspect the antibodies are generated against only the Fc region ofa foreign antibody. Essentially, the animal is immunized with only theFc region fragment of a foreign antibody, such as murine. The polyclonalantibodies are collected from subsequent bleeds, digested with anenzyme, pepsin or papain, to produce monovalent fragments. The fragmentsare then affinity purified on a column comprising whole immunoglobulinprotein that the animal was immunized against or just the Fc fragments.

Solid Supports

In an exemplary embodiment is provided dyes covalently conjugated to asolid support. This includes, but is not limited to, any combination ofcompounds disclosed above and any solid support disclosed herein.Typically the compound comprises a reactive group that facilitates thecovalent conjugation to the solid support. The reactive group maycontain both a reactive functional moiety and a linker, or only thereactive functional moiety.

Provided in one embodiment is a first composition that comprises apresent compound, a first fluorophore, and a solid support. Provided inanother embodiment is a second composition that includes a firstcomposition in combination with a second conjugate. The second conjugatecomprises a solid support or carrier molecule, as disclosed above, thatis covalently bonded to a second fluorophore. The first and secondfluorophore have different structures and preferably have differentemission spectra. Even more preferably, the first and secondfluorophores are selected so that their fluorescence emissionsessentially do not overlap. In another embodiment the fluorophores havedifferent excitation spectra, alternatively the fluorophores are excitedby the same laser.

The fluorophore on the second conjugate can include substantially anyfluorescent structure known in the art including, but not limited to,small organic fluorophores, fluorescent proteins, and reporter groupsthat are not necessarily fluorescent but which, under correctconditions, convert a fluorogenic substrate into a fluorophore, e.g.,horseradish peroxidase. Exemplary second fluorophores of use in thepresent invention include those that include a moiety that is a memberselected from a coumarin, a xanthene (e.g., fluorescein), a cyanine, apyrene, a borapolyazaindacene, an oxazine, and bimane.

The solid support (or carrier molecule) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various solid supports is generallyapplicable to this embodiment of the invention as well as otherembodiments.

A variety of solid supports are useful in the present invention. In anexemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound through the reactive group, if present, or through acarrier molecule, if present. In another exemplary embodiment, at leastone member selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, and R¹¹,comprises a solid support or is attached to a solid support. In oneaspect, at least one of R¹, R², R³, R⁴, or R⁵, comprises a solid supportor is attached to a solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

In another embodiment is provided a method for forming conjugates of thepresent dye compounds and a carrier molecule or solid support. Thismethod comprises:

-   -   a) combining a present compound with a carrier molecule or solid        support to form a combined sample, wherein the compound        comprises a reactive group; and,    -   b) incubating the combined sample for a sufficient amount of        time for the compound to form a covalent bond with either the        carrier molecule or solid support whereby a dye conjugate is        formed.

The dye conjugates of the carrier molecules or solid supports, e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivedyes of the invention and are generally prepared by means wellrecognized in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra,(2002)). Preferably, conjugation to form a covalent bond consists ofsimply mixing the reactive compounds of the present invention in asuitable solvent in which both the reactive compound and the substanceto be conjugated are soluble. The reaction preferably proceedsspontaneously without added reagents at room temperature or below. Forthose reactive compounds that are photoactivated, conjugation isfacilitated by illumination of the reaction mixture to activate thereactive compound. Chemical modification of water-insoluble substances,so that a desired compound-conjugate may be prepared, is preferablyperformed in an aprotic solvent such as dimethylformamide,dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform.Similar modification of water-soluble materials is readily accomplishedthrough the use of the instant reactive compounds to make them morereadily soluble in organic solvents.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about.1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive compoundis then dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of conjugation when added toa solution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of thecompound are added to the protein, the conjugate is chromatographicallypurified to separate unconjugated compound and the compounde-proteinconjugate is tested in its desired application.

Following addition of the reactive compound to the component solution,the mixture is incubated for a suitable period (typically about 1 hourat room temperature to several hours on ice), the excess compound isremoved by gel filtration, dialysis, HPLC, adsorption on an ion exchangeor hydrophobic polymer or other suitable means. The compound-conjugateis used in solution or lyophilized. In this way, suitable conjugates canbe prepared from antibodies, antibody fragments, avidins, lectins,enzymes, proteins A and G, cellular proteins, albumins, histones, growthfactors, hormones, and other proteins.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides) or multiple types of reactive sites(e.g. amines, thiols, alcohols, phenols) may be available, as is typicalfor proteins. Selectivity of labeling is best obtained by selection ofan appropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of compound istypically used, relative to the expected degree of compoundsubstitution. Any residual, unreacted compound or a compound hydrolysisproduct is typically removed by dialysis, chromatography orprecipitation. Presence of residual, unconjugated dye can be detected bythin layer chromatography using a solvent that elutes the dye away fromits conjugate. In all cases it is usually preferred that the reagents bekept as concentrated as practical so as to obtain adequate rates ofconjugation.

In an exemplary embodiment, the conjugate of the invention is associatedwith an additional substance, that binds either to the fluorophore orthe conjugated substance (carrier molecule or solid support) throughnoncovalent interaction. In another exemplary embodiment, the additionalsubstance is an antibody, an enzyme, a hapten, a lectin, a receptor, anoligonucleotide, a nucleic acid, a liposome, or a polymer. Theadditional substance is optionally used to probe for the location of thedye-conjugate, for example, as a means of enhancing the signal of thedye-conjugate.

Method of Use

The present invention also provides methods of using the compoundsdescribed herein to detect an analyte in a sample. Those of skill in theart will appreciate that this focus is for clarity of illustration anddoes not limit the scope of the methods in which the compounds of theinvention find use.

In certain embodiments, the compounds of the present invention areutilized to stain a sample to give a detectable optical response underdesired conditions by a) preparing a dye solution comprising a dyecompound described above, at a concentration sufficient to yield adetectable optical response under the desired conditions; combining thesample of interest with the dye solution for a period of time sufficientfor the dye compound to yield a detectable optical response under thedesired conditions; and c) illuminating the sample at a wavelengthselected to elicit the optical response. Optionally, the sample iswashed to remove residual, excess or unbound dye. The dye compoundtypically forms a covalent or non-covalent association or complex withan element of the sample, or is simply present within the bounds of thesample or portion of the sample.

In one embodiment, the staining is used to determine a specifiedcharacteristic of the sample by further comparing the optical responsewith a standard or expected response. For example, the dye solution isused to monitor specific components of the sample with respect to theirspatial and temporal distribution in the sample. Alternatively, the dyepreferentially binds to a specific analyte in a sample, enabling theresearcher to determine the presence or quantity of that specificanalyte. In this instance the dye is typically conjugated to a carriermolecule or solid support that directly interacts with the analyte ofinterest. In this way the dye of the present invention is a reportermolecule. However, in certain circumstances the dye may bind directly,covalently or no-covalently, to an analyte of interest in the sample.The desired analysis to be performed determines the composition of thedye solution and chemical nature of the dye itself. In another example,the dye is bound by an antibody directed against the dye itself,typically resulting in the fluorescence quenching of the dye.

In another embodiment of the invention, the dye compounds of theinvention possess utility as laser dyes according to methods known inthe art. In particular, the dyes are excited by a UV or violet laser.This is particularly advantages for multiplexing wherein many known dyesare excited by wavelengths at 488 nm or longer. Thus, in one embodimentthe present dyes are excited at about a 405 nm range but not at 488 nmor longer and their emission spectra is distinguishable from those dyesthat are excited at longer wavelengths. These dyes have particularapplications when analyzed by flow cytometry. In addition, these dyesalso find use in applications with other dyes that are excited at about400 nm because the long Stokes shift of the present compound allows forthe detectable signal to be resolved compared to other dyes. Thus, inone aspect is a dye with a Stokes shift longer than about 50 nm,preferably more than about 100 nm, even more preferable longer thanabout 150 nm. In one aspect the present compounds have a Stokes shift ofabout 140 nm.

In one aspect of the invention is provided a method for detecting ananalyte in a sample, wherein the method comprises:

-   -   a. combining a present compound with a sample to form a combined        sample;    -   b. incubating the combined sample for a sufficient amount of        time for the compound to associate with the analyte in the        sample to form an incubated sample;    -   c. illuminating the incubated sample with an appropriate        wavelength to form an illuminated sample; and    -   d. detecting the illuminated sample whereby the analyte in the        sample is detected.

The combined sample of the conjugate and the analyte is incubated underany appropriate conditions for a length of time sufficient for at leasta fraction of the analyte population to interact with the conjugate. Theinteraction can be by any known interaction mechanism, and the presentinvention is not limited to application with any single type ofanalyte-conjugate interaction mechanism. The interaction between theanalyte and the conjugate results in the formation of a fluorescentanalyte. The fluorescent analyte is readily detected and/or quantitatedby irradiating it with light of a wavelength that causes the fluorescentanalyte to emit fluorescence.

In one embodiment the dye is illuminated with a wavelength about 405 nm,common on violet lasers. In a particular aspect the illumination and/ordetecting step comprises a flow cytometer.

In the method described above, any number or combination ofpurification, separation or derivatization steps are optionally includedas steps in the method. In an exemplary embodiment, the fluorescentanalyte is separated from the remainder of the sample, fromnon-fluorescent analyte or from excess unbound conjugate prior todetermining the fluorescence of the fluorescent analyte.

In another exemplary embodiment, the invention provides a multicolormethod for detecting an analyte or more than one analyte. For example,when it is desired to detect, and particularly to confirm the identityof an analyte, more than one fluorescent conjugate, preferablyfluorescing at different wavelengths can be co-localized on the singleanalyte.

Therefore, a method for detecting a first analyte and a second analytein a sample is provided. The method includes incubating the sample witha composition of the invention that includes first and secondfluorescent labeled conjugates. The component of the first conjugate isa binding partner for the first analyte and the component of the secondconjugate is a binding partner for the second analyte. The incubationcontinues for a time and under conditions appropriate to induce aninteraction between at least a fraction of the population of the firstanalyte with the first conjugate. During this incubation period, it isgenerally preferred that a similar interaction occurs between the secondanalyte and second conjugate, however, it is within the scope of theinvention to change the incubation conditions as necessary to drive theformation of a conjugate-analyte complex between the second conjugateand second analyte.

Following the formation of at least the first analyte-conjugate complex,the sample is illuminated with light of a wavelength appropriate tocause the complex to fluoresce, thereby detecting the first analyte. Thesecond analyte is detected in a similar manner and may be detectedsimultaneously with the first analyte or by the sequential illuminationof the sample with wavelengths appropriate to induce the fluorescence ofeach fluorescent conjugate. In another embodiment the use of more thanone color of fluorescent conjugate per analyte provides assays in whichspecificity is dramatically increased, by requiring that the differentcolors or color combinations of the fluorescent conjugates coincidespatially during detection. This can dramatically reduce or eveneliminate the detection of nonspecifically bound targets or labels,enhancing specificity and sensitivity of the assay. Underlying theimprovement represented by the use of multiple differently coloredfluorescent conjugates is the improbability of accidentally encounteringtwo or more preselected different colors at the same location at thesame time. The improbability increases as more fluorescent conjugates ofdifferent colors are used. Alternatively, in another exemplaryembodiment, the emission from the two or more differently coloredfluorescent conjugates combines to form a third color, which is nototherwise present in the assay.

In an exemplary application of the present method, different features ofan analyte, e.g., a cell or epitopes of a molecule, are labeled withdifferent colored fluorescent conjugates. The target is detected and itsidentity is confirmed using the colocalization or “coincidence” of eachcolor on each target. Coincidence staining allows for the detection anddifferentiation of different organisms or strains of organismsexpressing different surface markers. Moreover, coincidence stainingprovides a method of distinguishing molecules of different structuredown to the level of isomeric differences and differences instereochemistry.

In the detection of pathogenesis, the most direct analyte is thepathogenic organism itself. In this case, assays preferably identifyparticular features of the organism such as surface proteins. To furtheraid in characterization, it is preferred to assay for molecular analytesas well. An example of a molecular analyte is an exotoxin such ascholera toxin. Antigen specific binding receptors are generated thatrecognize different characteristics of an analyte with high specificity.In the case of molecular analytes, receptors recognize differentepitopes of a protein or small molecule, while cellular analytes arerecognized through different molecules on the cell surface.

Although the fluorescence from each conjugate can be detectedsimultaneously, in one embodiment, to facilitate coincidence staining,the fluorescence from each analyte is detected independently.

In another exemplary embodiment, colocalization is used to differentiatebetween the formation of an analyte-conjugate complex and non-specificbinding of the analyte to another species within the assay system. Theintrinsic sensitivity of an assay often is limited by nonspecificbinding of the analyte or other assay mixture components to thesubstrate. Single analyte coincidence staining can be used todifferentiate between specific binding of the analyte to the conjugateand non-specific binding of assay mixture components to the conjugatebased on the colocalization of fluorescent conjugate colors. Those ofskill in the art will appreciate that coincidence staining as describedherein is useful to distinguish nonspecific binding in both solid-phase(e.g., gene chip) and solution-based assays.

Coincidence staining can also be used to identify a single analyte. Forexample, one may wish to confirm the presence of a selected analyte in amixture of analytes that are structurally similar (e.g. having a commonepitope) or that have similar affinity for the component of theconjugate. In such circumstances, it may prove that the detection of asingle epitope is not sufficient for conclusive identification of atarget. Measuring the level of 2, preferably 3, more preferably 4 andeven more preferably 5 or more markers within a single analyte, providesan unambiguous profile specific for the analtye of interest.

In another exemplary embodiment, the present invention provides methodsfor evaluating cell viability. In this instance the present compoundsthat comprise an amine reactive group, meaning a reactive group thatwill form a covalent bond with an amine on a solid support or carriermolecule, are incubated with a sample comprising both live and deadcells. The present compounds will for conjugates with the amine groupson the membrane of live cells producing a fluorescent signal whenilluminated with an appropriate light source. However, for dead cellsthe cell membrane has been compromised allowing entry of the compoundsinto the cells where they form conjugates with the amines in the cells.These conjugates in the dead cells produce a fluorescent signal, whenilluminated with appropriate light source, that is significantlybrighter than the signal from live cells. In one aspect the signal fromdead cells is 10× brighter than the signal from live cells. In anotheraspect the signal from dead cells is 50× brighter than the signal fromlive cells. In yet another aspect the signal from dead cells is 100×brighter than the signal from live cells. See Example 37 and 38.

Thus, in one embodiment the present invention provides a method forevaluating the viability of cells in a sample, comprising

-   -   a. combining a present compound with a sample to form a combined        sample, wherein the compound comprises an amine reactive group;    -   b. incubating the combined sample for a sufficient amount of        time for the compound to form a covalent bond with amine groups        on a cell membrane of live cells and to form covalent bonds with        amine groups inside dead cells in the sample to form an        incubated sample;    -   c. illuminating the incubated sample with an appropriate        wavelength to form an illuminated sample; and    -   d. detecting the illuminated sample whereby the via of cells in        a sample is evaluated.

In an alternative embodiment, dead cells are stained with a presentcompound and live cells stained with a know live cell stain wherein thetwo fluorescent signals can be distinguished from each other.

In another exemplary embodiment, the present invention provides a methodfor distinguishing between organisms expressing the same surfacemarkers. Using coincidence staining, it is possible to identifydifferences in targets based on the ratio of surface marker expression.For example, despite intense efforts, no single binding-receptor hasbeen found for the unambiguous detection of B. anthracis spores, due toextensive cross-reactivity with related B. cereus and B. thuringiensis,which are genetically a single species (Helgason et al., Appl. Envir.Microbiol. 66:2627-2630 (2000)). Despite being of the same species,however, the relative amount of various surface proteins is differentbetween the three bacilli. Thus, multi-point detection of a variety ofmarkers at the single cell level will provide the specificity requiredto detect B. anthracis.

Diagnostic tests that detect the presence or absence of a single markerare unable to distinguish among strains that are nearly identical at thegenetic level, highlighting the need for new tools to distinguishbetween closely related organisms. Epidemics caused by emerging variantsof known pathogens are a common theme in infectious diseases (Jiang etal., Appl Environ Microbil 66:148-53 (2000)) (Hedelberg et al. Nature406:477-483 (2000)). There is also the problem of deliberate engineeringof pathogens, incorporating virulence determinants from other species.An attack by such pathogens would be misdiagnosed due to the presence ofmarkers not normally found in the attacking pathogen. By probingmultiple markers within a single organism, using the methods of theinvention, such variants are detected and preferably identified.

Detection by eye is also useful in those embodiments of the inventionrelying on coincidence staining. The human eye is extremely good atdistinguishing between subtly different combinations of colors,especially when the colors are chosen correctly. By way of illustration,it is trivial for people to distinguish between the colors red, greenand yellow. Yellow, however, is simply the spectral sum of red andgreen, so if red and green fluorescent conjugates are used forcoincidence staining, positive assay signal can easily be identified bythe perceived color, yellow. Other color combinations of use in thisembodiment of the invention will be readily apparent to those of skillin the art, such as combinations of red, green and blue to form white.

Solutions of the compounds of the invention are prepared according tomethods generally known in the art. As with related known fluorophoresand fluorogens, the dyes and dye-conjugates are generally soluble inwater and aqueous solutions having a pH greater than or equal to about6. Stock solutions of pure dyes, however, are typically dissolved inorganic solvent before diluting into aqueous solution or buffer.Preferred organic solvents are aprotic polar solvents such as DMSO, DMF,N-methylpyrrolidone, acetone, acetonitrile, dioxane, tetrahydrofuran andother nonhydroxylic, completely water-miscible solvents. In general, theamount of dye or conjugate in the dye solution is the minimum amountrequired to yield detectable staining in the sample within a reasonabletime, with minimal background fluorescence or undesireable staining. Theexact concentration of dye or dye-conjugate to be used is dependent uponthe experimental conditions and the desired results, and optimization ofexperimental conditions is typically required to determine the bestconcentration of stain to be used in a given application. Theconcentration of dye present in the dye solution typically ranges fromnanomolar to micromolar. The required concentration for the dye solutionis determined by systematic variation in dye or dye-conjugateconcentration until satisfactory dye staining is accomplished. Thestarting ranges are readily determined from methods known in the art foruse of similar compounds under comparable conditions for the desiredoptical response.

The dye compounds are advantageously used to stain biological samples,i.e. samples that comprise biological components. In one embodiment ofthe invention, the sample comprises heterogeneous mixtures ofcomponents, including intact cells, cell extracts, bacteria, viruses,organelles, and mixtures thereof. In another aspect of the invention,the sample comprises a single component or homogeneous group ofcomponents, e.g. biological polymers such as amino acid polymers,nucleic acid polymers or carbohydrate polymers, or lipid membranecomplexes, whether the polymers are synthetic or natural.

The sample is typically stained by passive means, i.e., by incubationwith the dye solution. Any other method of introducing the dye into thesample, such as microinjection of a dye solution into a cell ororganelle, can be used to accelerate introduction of the dye into thesample. The dyes of the present invention are generally non-toxic toliving cells and other biological components, within the concentrationsof use.

In one aspect of the invention, the sample is obtained directly from aliquid source or as a wash from a solid material (organic or inorganic)or a growth medium in which cells have been introduced for culturing, ora buffer solution in which cells have been placed for evaluation. In afurther aspect, cells are isolated from biological fluids such as blood.Where the sample comprises cells, the cells are optionally single cells,including microorganisms, or multiple cells associated with other cellsin two or three dimensional layers, including multicellular organisms,embryos, tissues, biopsies, filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot.

In yet another embodiment, the sample is present on or in solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane. In another aspect, the matrix is an electrophoretic gel, suchas is used for separating and characterizing nucleic acids or proteins.In another aspect, the matrix is a silicon chip or glass slide, and theanalyte of interest has been immobilized on the chip or slide in anarray. In yet another aspect, the matrix is a microwell plate ormicrofluidic chip, and the sample is analyzed by automated methods,typically by various methods of high-throughput screening, such as drugscreening.

The sample can be observed immediately after staining. A detectableoptical response means a change in, or occurrence of, an optical signalthat is detectable either by observation or instrumentally. Typicallythe detectable response is a change in fluorescence, such as a change inthe intensity, excitation or emission wavelength distribution offluorescence, fluorescence lifetime, fluorescence polarization, or acombination thereof. The degree and/or location of staining, comparedwith a standard or expected response, indicates whether and to whatdegree the sample possesses a given characteristic.

The sample is optionally combined with other solutions in the course ofstaining, including wash solutions, permeabilization and/or fixationsolutions, and other solutions containing additional detection reagents.Washing following staining generally improves the detection of theoptical response due to the decrease in non-specific backgroundfluorescence after washing. Satisfactory visualization is possiblewithout washing by using lower labeling concentrations. A number offixatives and fixation conditions suitable for practicing this inventionare known in the art, including formaldehyde, paraformaldehyde,formalin, glutaraldehyde, cold methanol and 3:1 methanol:acetic acid.Fixation is typically used to preserve cellular morphology and to reducebiohazards when working with pathogenic samples. Selected embodiments ofthe dyes described above are well retained in cells, and sample cellsstained with these dyes retain considerable fluorescent staining afterfixation. Fixation is optionally followed or accompanied bypermeabilization, such as with acetone, ethanol, DMSO or variousdetergents, to allow bulky dye compounds, including dye-conjugatesdescribed above, to cross cell membranes, according to methods generallyknown in the art. The staining of the present invention is optionallycombined with the use of an additional detection reagent that produces adetectable response due to the presence of a specific cell component,intracellular substance, or cellular condition, according to methodsgenerally known in the art. Where the additional detection reagent hasspectral properties that differ from those of the subject dye compounds,multi-color applications are possible. The compounds of the inventionare also of use to derivative low molecular weight compounds for theiranalysis by capillary zone electrophoresis (CZE), HPLC or otherseparation techniques.

Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared. The sample includes, without limitation,any biological derived material or aqueous solution that is believed tocontain a target analyte or ligand. Alternatively, samples also includematerial in which an analyte or ligand has been added.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein an analyte of interest has been secretedinto the medium. Alternatively, the sample may be whole organs, tissueor cells from the animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammaryglands, mesothelium, and the like. Cells include without limitationprokaryotic cells and eukaryotic cells that include primary cultures andimmortalized cell lines. Eukaryotic cells include without limitationovary cells, epithelial cells, circulating immune cells, β cells,hepatocytes, and neurons.

One may use an individual compound of the invention, multiple compoundsof the invention or a combination of a compound of the invention and afluorophore or quencher of a different structure in order to detect thepresence of or determine the characteristics of a target in a sample.

When the components of the invention are species that bind to targetsthat are specific biological structures (e.g, enzymes, receptors,ligands, antigens, antibodies, etc.), the reaction time between thecompound or conjugate of the invention and the target will usually be atleast about 5 min, more usually at least about 30 min and preferably notmore than about 180 min, preferably not more than about 120 min,depending upon the temperature, concentrations of enzyme and substrate,etc. By using a specific time period for the reaction or taking aliquotsat 2 different times, the rate of reaction can be determined forcomparison with other determinations. The temperature will generally bein the range of about 20 to 50° C., more usually in the range of about25 to 40° C.

Various buffers can be used in the assays of the invention. Thesebuffers include PBS, Tris, MOPS, HEPES, phosphate, etc. The pH will varydepending upon the particular assay system, generally within a readilydeterminable range wherein one or more of the sulfonic acid moieties isdeprotonated. The concentration of buffer is generally in the range ofabout 0.1 to 50 mM, more usually 0.5 to 20 mM.

In many instances, it may be advantageous to add a small amount of anon-ionic detergent to the sample. Generally the detergent will bepresent in from about 0.01 to 0.1 vol. %. Illustrative non-ionicdetergents include the polyoxyalkylene diols, e.g. Pluronics, Tweens,Triton X-100, etc.

In fluorescence experiments, the reaction is optionally quenched.Various quenching agents may be used, both physical and chemical.Conveniently, a small amount of a water-soluble solvent may be added,such as acetonitrile, DMSO, SDS, methanol, DMF, etc.

Illumination

The compounds of the invention may, at any time after or during anassay, be illuminated with a wavelength of light that results in adetectable optical response, and observed with a means for detecting theoptical response. Upon illumination, such as by an violet or visiblewavelength emission lamp, an arc lamp, a laser, or even sunlight orordinary room light, the fluorescent compounds, including those bound tothe complementary specific binding pair member, display intense visibleabsorption as well as fluorescence emission. Selected equipment that isuseful for illuminating the fluorescent compounds of the inventionincludes, but is not limited to, hand-held ultraviolet lamps, mercuryarc lamps, xenon lamps, argon lasers, laser diodes, and YAG lasers.These illumination sources are optionally integrated into laserscanners, flow cytometer, fluorescence microplate readers, standard ormini fluorometers, or chromatographic detectors. This fluorescenceemission is optionally detected by visual inspection, or by use of anyof the following devices: CCD cameras, video cameras, photographic film,laser scanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. Where the sample is examined using a flowcytometer, a fluorescence microscope or a fluorometer, the instrument isoptionally used to distinguish and discriminate between the fluorescentcompounds of the invention and a second fluorophore with detectablydifferent optical properties, typically by distinguishing thefluorescence response of the fluorescent compounds of the invention fromthat of the second fluorophore. Where a sample is examined using a flowcytometer, examination of the sample optionally includes isolation ofparticles within the sample based on the fluorescence response by usinga sorting device.

Kits of the Invention

In another aspect, the present invention provides kits that include afluorescent compound of the invention. The kit will generally alsoinclude instructions for using the compound of the invention in one ormore methods.

In an exemplary embodiment, the kit includes a reactive compound of theinvention and instructions for conjugating the dye to any substancepossessing an appropriate functional group, and optionally forrecovering or purifying the materials labeled thereby. This combinationof reactive dye and instructions therefore comprise a kit for labelingan appropriate substance. Selected appropriate substances include, butare not limited to, polymers of biological molecules (e.g. proteins,oligonucleotides or carbohydrates), polymeric resins and plastics (e.g.polystyrene), metals, glasses, and other organic or inorganicsubstances. The dyes of the present invention are well-suited for thepreparation of such a kit.

In another exemplary kit of the invention, the instructions provided arefor performing an assay that detects an analyte or ligand in a sample.Typically the kit would cpmrpirse a dye conjugated to a secondaryantibody. In this way the end user would provide the primary antibodyresulting in an unlimited number of ligand or analytes that could bedetected. Alternatively the kit would comprise the dye-labeled secondaryand a primary antibody. For example, in one embodiment, directions areprovided for detecting a cell receptors, or an enzyme, organism, orother ligands that are bound by antibodies.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Synthesis of Compound 1

A mixture of 3.6 g of 4-hydroxyacetophenone, 5.7 g of methyl5-bromovalerate, and 8.1 g of K₂CO₃ in 50 mL of DMF was heated at 75° C.overnight. After the reaction mixture was cooled to room temperature, itwas extracted with ethyl acetate, washed with water, and dried overanhydrous Na₂SO₄ to obtain product Compound 1.

Example 2 Synthesis of Compound 2

To 7.0 g of Compound 1 in 60 mL of CHCl₃, 4.3 g of bromine (in 10 mL ofCHCl₃) was added slowly at room temperature and stirred for anadditional 3 h. All volatile components were removed under reducedpressure and the residue was purified on a silica gel column with ethylacetate and hexanes.

Example 3 Synthesis of Compound 3

To 6.0 g of Compound 2 in 30 mL of CHCl₃ at 0° C., 3.1 g of metheamine(in 50 mL of CHCl₃) was added slowly at room temperature and stirred foranother 4 h. All volatile components were removed under reduced pressureto give the product Compound 3.

Example 4 Synthesis of Compound 4

To 9.2 g of Compound 3 suspended in 150 mL of ethanol at 0° C., 12 mL ofconcentrated HCl was added slowly and the mixture was stirred at 40° C.for 4 h. All volatile components were then removed under reducedpressure to recover product Compound 4.

Example 5 Synthesis of Compound 5

To 9.0 g of Compound 4 in 150 mL of CHCl₃ at 0° C., 12 g oftriethylamine was introduced followed by a solution ofiso-nicotinoylchloride hydrochloride (6.4 g in a mixture of 50 mL ofCHCl₃ and 50 mL of DMF). The mixture was warmed to room temperature andstirred at room temperature overnight. The reaction mixture was thenpoured into water and extracted with CHCl₃. The organic layer was washedwith saturated aqueous Na₂CO₃ and water and the crude product waspurified on a silica gel column with MeOH and CHCl₃.

Example 6 Synthesis of Compound 6

A mixture of 2.0 g of Compound 5 and 25 mL of phosphorous oxychloridewas heated at reflux temperature for 10 h. After the reaction mixturewas cooled to room temperature, excess phosphorous oxychloride wasremoved by rotary evaporation and the residue was dissolved in 100 mL ofethyl acetate and washed with saturated aqueous Na₂CO₃, water and driedover anhydrous Na₂SO₄. The solvent was removed to give 1.9 g of productCompound 6.

Example 7 Synthesis of Compound 7

A mixture of compound 1.9 g of Compound 6 and 8.0 g of propane sultonein 30 mL of acetonitrile was heated at 110° C. for 2 h. After cooling toabout 50° C., an ethyl acetate/hexanes mixture (150 mL, 1:1, v/v) wasadded and product Compound 7 was collected by filtration.

Example 8 Synthesis of Compound 8

A solution of 3.6 g of Compound 7 in 150 mL of concentrated HCl and 100mL of water was heated at 65° C. for 2 days and all volatile componentswere removed under reduced pressure to give the crude carboxylic acid.The crude acid was dissolved in 15 mL concentrated H₂SO₄ and aftercooled to 0° C., 15 mL of 30% fuming H₂SO₄ was added and the mixture wasstirred at the low temperature for 1 h. The reaction mixture was pouredinto 500 mL of cold Et₂O and the crude product was collected byfiltration and further purified by HPLC to give 1.6 g of pure productCompound 8.

Example 9 Synthesis of Compound 9A

To 1.6 g of Compound 8 in 500 mL of anhydrous DMA (500 mL), 1.6 g of N,N′-disuccinimidyl carbonate and 62 mg of N,N-dimethylaminopyridine wereadded and the reaction mixture was stirred at room temperature as asuspension for 2 days. At the end of the period, the volume of thereaction mixture was reduced to ˜100 mL under reduced pressure and 300mL of ethyl acetate was added and stirred for ½ h. The solid wascollected by filtration and washed with ethyl acetate to give 1.7 g ofproduct Compound 9A.

Example 10 Synthesis of Compound 9B

To 100 mg of Compound 9A in 10 mL of anhydrous dimethylacetamide at 0°C., 100 mg of hydrazine was added and the reaction mixture was stirredat 0° C. for 1 h. The solvent was removed under vacuum and the residuewas purified by HPLC to give product Compound 9B

Example 11 Synthesis of Compound 9C

To 100 mg of Compound 9A in 10 mL of anhydrous dimethylacetamide at 0°C., 90 mg of N-(2-aminoethyl)maleimide trifluoroacetic acid salt and 0.1mL of triethyl amine were added and the reaction mixture was stirred at0° C. for 1 h. The solvent was removed under vacuum the residue waspurified by HPLC to give product Compound 9C.

Example 12 Synthesis of Compound 10

Compound 10 was prepared by following the same procedure for compound 1with 4-hydroxyphenylacetone as the starting material.

Example 13 Synthesis of Compound 11

A mixture of 2.5 g of Compound 10, 0.72 g of hydroxylaminehydrochloride, 1.8 g of diisopropylethylamine in 50 mL of EtOH washeated at 75° C. for 20 minutes. The solvent was removed and the residuewas dissolved in 200 mL of ethyl acetate and washed with 1% HCl, anddried over anhydrous Na₂SO₄ to give the product Compound 11.

Example 14 Synthesis of Compound 12

A mixture of 1.9 g of Compound 11 and 1.8 g of iso-nicotinoylchloridehydrochloride in 30 mL of pyridine was heated the at 95° C. overnight.The solvent was removed and the residue was partitioned between ethylacetate and saturated sodium carbonate and the organic layer was driedover anhydrous Na₂SO₄. The crude product is purified on a silica gelcolumn with MeOH and CHCl₃.

Example 15 Synthesis of Compound 13

Compound 13 was prepared from Compound 12 by following the sameprocedures for the transformation of Compound 6 to compound 9A.

Example 16 Synthesis of Compound 14

A mixture of 300 mg of 2-amino-4′-methoxyacetonphenone hydrochloride and270 mg of 4-quinolinecarboxylic acid in 5 mL of phosphorous oxychloridewas refluxed for 5 h and after cooling to room temperature, excessphosphorous oxychloride was evaporated under reduced pressure. Theresidue was partitioned ethyl acetate and saturated sodium carbonate andthe organic layer was then washed with water and dried over anhydrousNa₂SO₄. The crude product is purified on a silica gel column with CHCl₃and ethyl acetate.

Example 17 Synthesis of Compound 15

Compound 15 was prepared by following the same procedures for compound 8using compound 14 as starting material.

Example 18 Synthesis of Compound 16

Compound 16 was prepared from 4-chloroacetyl-1-methoxynaphthalene byfollowing the aforementioned procedures.

Example 19 Synthesis of Compound 17

Compound 17 was prepared from 2, 4-dimethoxyacetophenone and6-methoxy-2-phenyl-4-quinolinecarboxylic acid by following theaforementioned procedures.

Example 20 Synthesis of Compound 18

Compound 18 was prepared from 2, 4-dimethoxyacetophenone and4-quinolinecarboxylic acid by following the aforementioned procedures.

Example 21 Synthesis of Compound 19

Compound 19 was prepared from 3, 4-dimethoxyacetophenone and4-quinolinecarboxylic acid by following the aforementioned procedures.

Example 22 Synthesis of Compound 20

A mixture of 2.5 g of 6′-methoxy-2′-acetonaphthone and 5.6 g of copper(II) bromide in 100 mL of CHCl₃/ethyl acetate (1:1 v/v) was heated atreflux temperature for 3 h. After the reaction mixture was cooled toroom temperature, the precipitate was filtered off and washed withCHCl₃. The crude product was recrystallized from CHCl₃ and hexanes.

Example 23 Synthesis of Compound 21

To 0.7 g of compound 20 in 50 mL of CHCl₃ at 0° C., 0.44 g of metheaminewas added slowly at room temperature and stirred overnight. At the endof the period 100 mL of hexane was added to the reaction mixture and theprecipitate was collected to give the product Compound 21 (1.0 g).

Example 24 Synthesis of Compound 22

To 0.65 g of Compound 21 suspended in 30 mL of ethanol at 0° C., 5 mL ofconcentrated HCl was added slowly and the mixture was stirred at 40° C.for 4 h. All volatile components were then removed under reducedpressure to recover product Compound 22.

Example 25 Synthesis of Compound 23

A mixture of 100 mg of Compound 22 and 150 mg of 4-quinolinecarboxylicacid in 5 mL of phosphorous oxychloride was refluxed for 10 h and aftercooling to room temperature, excess phosphorous oxychloride wasevaporated under reduced pressure. The residue was partitioned withethyl acetate and saturated sodium carbonate and the organic layer wasthen washed with water and dried over anhydrous Na₂SO₄. The crudeproduct is purified on a silica gel column with CHCl₃ and ethyl acetate.

Example 26 Synthesis of Compounds 24A and 24B

Compounds 24A and 24B were prepared from Compound 23 by first reactingwith propane sultone followed by sulfonation with fuming sulfuric acidaccording to the aforementioned procedures.

TABLE 3 Spectral properties of selected compounds of the invention inPBS buffer (pH = 7.0) Absorbance maximum Emission maximum Compound (nm)(nm) Compound 8 396 549 Compound 13 404 550 Compound 15 428 597 Compound18 451 632 Compound 19 401 462

Example 27 Synthesis of Compounds 25-31

To a mixture of 4-hydroxyphenylboronic acid (1.38 g), 2,5-dibromothiophene (4.84 g), tetrakis(triphenylphosphine)palladium (0)(1.24 g), toluene (60 mL) and iso-propyl alcohol (30 mL), 4.14 g ofK₂CO₃ (in 10 mL of water) was added. N₂ was bubbled through the mixturefor several minutes and the resulting suspension was heated at 100° C.under N₂ with vigorous stirring for 5 h. After cooling to roomtemperature and diluting with water, the resulting mixture was acidifiedto pH 2 with 4% HCl and extracted with EtOAc. The organic layer waswashed with water and dried over anhydrous Na₂SO₄. The residue waspurified on a silica gel column with hexanes and EtOAc to give 1.2 g of25.

Compound 25 (1.2 g) was heated in 30 mL of DMF with methyl5-bromovalerate (1.38 g) and K₂CO₃ (1.95 g) at 75° C. for 4 h. Themixture was cooled to room temperature, diluted with water and extractedwith EtOAc. The crude was purified on a silica gel column with hexanesand EtOAc to give 1.25 g of 26.

To a mixture of compound 26 (1.05 g), 4-pyridineboronic acid (0.53 g),tetrakis(triphenylphosphine)palladium (0) (0.35 g), toluene (30 mL) andiso-propyl alcohol (15 mL), 1.2 g of K₂CO₃ (in 5 mL of water) was added.The suspension was purged with N₂ and heated at 100° C. under N₂ for 2h. The reaction mixture was cooled to room temperature, diluted withwater and extracted with EtOAc. The crude was purified on a silica gelcolumn with CHCl₃ and MeOH to give 0.81 g of 27.

A mixture of compound 27 (0.52 g) and 1, 3-propane sultone (4.0 g) wasrefluxed in 10 mL of CH₃CN for 10 h. After the mixture was cooled toabout 50° C., 150 mL of a 1/1 mixture (v/v) of EtOAc/hexanes was added,and product 28 was collected by filtration.

Compound 28 (0.36 g) was hydrolyzed with aqueous HCl (con. HCl/water, 60mL/20 mL) at 65° C. for 3 days. All volatile components were removed invacuo to obtain 29. The crude compound 29 thus obtained was dissolved inconc. H₂SO₄ (10 mL), cooled to 0° C. before 5 mL of 30% fuming H₂SO₄ wasintroduced slowly and the resulting mixture was stirred at 0° C. for 1h. The reaction mixture was then poured into 300 mL of cold Et₂O, andthe crude was collected by filtration and purified on HPLC to give 0.15g of 30.

To compound 30 (50 mg) in 10 mL of anhydrous dimethylacetamide, 40 mg ofN, N′-disuccinimidyl carbonate and 3 mg of 4-dimethylaminopyridine wereadded and the resulting mixture was stirred at room temperature for 3 h.At the end of the period, 100 mL of EtOAc was added and after stirringfor an additional 0.5 h, the solid was filtered, washed with EtOAc anddried in vacuo to give 50 mg of product 31.

Example 28 Synthesis of Compounds 32-34

To 2-amino-4′-methoxyacetonphenone hydrochloride (0.5 g) in 30 mL ofanhydrous dimethylacetamide at 0° C., triethylamine (3.0 mL) was addedand followed by iso-nicotinoylchloride hydrochloride (0.52 g). Themixture was then warmed to room temperature and stirred for anadditional 3 h. The reaction was diluted with water, extracted withEtOAc and dried over anhydrous Na₂SO₄. Crude yield was 0.63 g ofCompound 32.

A mixture of crude 32 (0.63 g) and Lawesson's reagent (2.4 g) wasrefluxed in 80 mL of anhydrous THF under N₂ for 8 h. The volatilecomponents were removed under reduced pressure and the crude waspurified on a silica gel column with CHCl₃ and MeOH to give 0.75 g of33.

A mixture of compound 33 (0.70 g) and 1,3-propane sultone (3.0 g) wasrefluxed in 10 mL of CH₃CN for 10 h. The mixture was cooled to ˜50° C.and 150 mL of a 1:1 (v/v) mixture of EtOAc/hexanes was added and theproduct, Compound 34, was collected by filtration.

Example 29 Synthesis of Compound 35

Compound 35 was prepared by following the same procedure for compound 30starting with compound 34.

Example 30 Synthesis of Compound 36

To N-[2-keto-2-(4-pyridyl)ethyl]-4-methoxybenzamide (0.50 g) in a flask,POCl₃ (10 mL) was added and refluxed for 1 h. The reaction mixture wascooled to room temperature and all volatiles were removed under reducedpressure and the residue was dissolved in EtOAc, and washed withsaturated aqueous Na₂CO₃. The crude thus obtained was purified on asilica gel column with CHCl₃ and MeOH to give 0.3 g of 36.

Example 31 Synthesis of Compound 37

Compound 37 was prepared by following the same procedure for compound 35starting from compound 36.

Example 32 Two Color Immunophenotyping Using Pacific Blue Dye andCompound 9

Human mononuclear cells were harvested from CPT (Ficol) tubes (BDBioscience). Cells were washed with 1% BSA/PBS and resuspended at aconcentration of 10 million/mL. A mouse anti-human CD4 primary antibody(BD Pharmingen) was labeled with the Zenon Pacific Blue™ Labeling Kit.The cells were then stained with Zenon Pacific Blue mouse anti-human CD4complex and 1 microgram of mouse anti-human CD8 Compound 9 for 30minutes. The cells were washed with 1% BSA/PBS, centrifuged, andresuspended with 400 microliters of 1% BSA/PBS. The samples wereanalyzed on a LSR II flow cytometer exiting with a 405 nm diode laser,collecting emission with 450/50 nm and 575/26 emission filters. Using aforward scatter vs. side scatter dot plot the lymphocytes were gated andplots were made using a lymphocyte gate. Compensation was applied usingsingle color controls. See, FIG. 1.

Example 33

Conjugation of Compound 9A Succinimidyl Ester (SE) and goat anti-mouseIgG (GAM) 0.221 mL (1.5 mg) of a 6.8 mg/mL solution of GAM in 10 mMpotassium phosphate, 150 mM sodium chloride buffer (PBS) was measuredinto test tubes and the pH raised to >8.0 with 22 μL 1 M sodiumbicarbonate, pH 9.0. The GAM solution was reacted with a 10, 20, 30, 40,or 50-fold molar excess of the Compound 9 at 20 mg/mL in anhydrous DMSOfor 1 h at RT. The dye-protein conjugates were separated from free dyeby size exclusion chromatography using 5-0.75×20 cm columns packed withBioRad™ Bio-Gel® P-30 fine in PBS and eluted with same. The initialprotein-containing band from each column was collected.

Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV/Visspectrometer. The fluorescence emission spectra were obtained using anAminco Bowman Series 2 Luminescence Spectrometer, excited at 390 nm.Cell staining of the GAM-Compound 9A conjugates was performed usingINOVA Diagnostics, Inc. prefixed HEp-2 cells in a 12 wells/slide formatto detect mouse anti-human IgG antibody labeling of human anti-nuclearantibodies. Samples were prepared in 1% bovine serum albumin in PBS at 1μg/mL and 10 μg/mL and let incubate 30 min at RT in both positive andcontrol wells. Wells were washed 4× with PBS, soaking for 10 minutes inPBS prior to wet mounting in PBS with a coverslip. The cells were imagedusing filter sets from Omega® Optical, XF12 in a Nikon Eclipse E400fluorescence microscope and Princeton Instruments, Inc. RTE/CCD-728-Ycamera using MetaMorph Imaging System from Universal ImagingCorporation.

Example 34

Conjugation of Compound 9A Succinimidyl Ester (SE) and streptavidin (SA)0.1 mL (1 mg) of a 10.0 mg/mL solution of SA in 0.1M sodium bicarbonatewas reacted with a 5, 10, 20, 30, 40, or 80-fold molar excess of theCompound 9A at 20 mg/mL in anhydrous DMSO for 1 h at RT. The dye-proteinconjugates were separated from free dye by size exclusion chromatographyusing 6-0.75×20 cm columns packed with BioRad™ Bio-Gel® P-30 fine in PBSand eluted with same. The initial protein-containing band from eachcolumn was collected.

Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV/Visspectrometer. The fluorescence emission spectra were obtained using anAminco Bowman Series 2 Luminescence Spectrometer, excited at 390 nm.Cell staining of the SA-Compound 9A conjugates was performed using INOVADiagnostics, Inc. prefixed HEp-2 cells in a 12 wells/slide format todetect biotinylated goat anti-human IgG antibody labeling of humananti-nuclear antibodies. Samples were prepared in 1% bovine serumalbumin in PBS at 10 μg/mL and let incubate 30 min at RT in bothpositive and control wells. Wells were washed 4× with PBS, soaking for10 minutes in PBS prior to wet mounting in PBS with a coverslip. Thecells were imaged using filter sets from Omega® Optical, XF12 in a NikonEclipse E400 fluorescence microscope and Princeton Instruments, Inc.RTE/CCD-728-Y camera using MetaMorph Imaging System from UniversalImaging Corporation.

Example 35 Comparison of Cascade Yellow and Compound 9a Conjugates

Mononuclear cells were harvested from CPT Ficoll tubes. Cells werecounted and resuspended at 1×10⁷/mL. 100 μL of cells were aliqouted totest tubes. Cells were blocked with 10 μL mouse IgG, incubated withappropriate primary antibody mouse anti-human CD4 biotin, RT/dark, 30min, cells were washed with 1% BSA/PBS, incubated with streptavidinconjugates at concentrations ranging from 1 μg-0.008 μg. Cells werewashed and resuspended in 1% BSA/PBS and analyzed on LSR II usingblue/red/violet/and UV excitation.

Results: Compound 9A is brighter than Cascade yellow and Compound 9Adoes not have 488 emission and has very little emission in the PacificBlue filter off of the violet. See FIG. 3.

Example 36 Comparison of Compound 9a Mouse Anti-Human CD8 to AmCyanMouse Anti-Human CD8 and CD4

Human PBMC's were harvested from the flow-through fraction of humanapheresis. Cells were resuspended at a concentration of 10E6 per ml inPBS containing 2% FBS and 0.1% sodium azide at 4° C. Cells were stainedwith mouse anti-human CD8 Compound 9A at a concentration of 2 ug/sampleor mouse anti-human CD8 AmCyan or mouse anti-human CD4 AmCyan andincubated for 30 minutes. The cells were washed, resuspended andanalyzed on a LSRII flow cytometer (BD Bioscience) using a 405 nm violetdiode laser (25 mW) and 530/30 or 560/55 nm bandpass filter. The resultsshowed Compound 9A anti-hCD8 was brighter than AmCyan anti-hCD8 andAmCyan anti-hCD4. See, FIG. 4A through FIG. 4H.

Example 37 Staining of Dead Cells and Fixed Cells with Compound 31

Using four populations of Jurkat cells: live culture cells, aged culturecells, heat-killed cells, and ETOH killed cells, each cell type issuspended at 1×10⁶ cells/ml in PBS. One ml of cell suspension was addedto a tube, and 5 μl of a 0.1 mg/ml solution of Compound 31 was added,mixed, and incubated 30 minutes at room temperature protected fromlight. After incubation, tubes were washed once with PBS, cells werepelleted, and pellet resuspended in one ml PBS. A BD LSRII flowcytometer was used to collect 10,000 events/tube, using 405 nmexcitation and 525/50 bandpass. After initial collection, cells wereresuspended in 3.7% formaldehyde in PBS, incubated 15 minutes, andre-run on the LSRII.

Live cells were distinguished from dead cells, with the live cellsshowing fluorescence slightly above autofluorescence and dead cellsshowing a fluorescence increase of at least one log unit. Using the MFI(Median fluorescent intensity) value of the live cells (MFI=108) andcalculating a MFI ratio for each cell type to calculate a Dead:Liveratio, a ratio of 35 was obtained with aged culture cells, a ratio of 92was obtained with ETOH-killed cells, and a ratio of 74 was obtained withheat-killed cells. After fixation, no changes in light scatter orfluorescence were noted. Compound 31 stains positive for dead cells andfixation with 3.7% formaldehyde does not alter results. See, FIG. 5Athrough FIG. 5D.

Example 38 Staining of Dead Cells with Compound 31 and Live Cells withTwo Different Calcein AM Stains

A mixture of live and heat-killed Jurkat cells and a mixture of live andethanol-killed Jurkat cells each were suspended at 1×106 cells/ml inPBS. One ml of cell suspension was added to a tube, and 5 μl of a 0.1mg/ml solution of Compound 31 was added to each set. To one set, calceingreen, AM was also added at 50 μM final concentration for dual colorstaining. To the other set of tubes, calcein violet, AM (InvitrogenCorp. C34858) was added for dual color staining. Tubes were incubated 30minutes at room temperature protected from light. After incubation,tubes were washed once with PBS, cells were pelleted, and the cellpellet resuspended in one ml PBS. A BD LSRII flow cytometer was used tocollect 10,000 events/tube, using 405 nm excitation and 525/50 bandpassfor Compound 31, 405 nm excitation with 450/50 bandpass fro the calceinviolet, AM stain, and 488 nm excitation with 530/30 bandpass for thecalcein green, AM stain. Single and dual color fluorescence wascollected.

Live cells (as defined by the calcein green, AM and the calcein violet,AM staining) and dead cells (as defined by Compound 31) were easilydistinguished as separate and mutually exclusive populations. Compound31 stains positive for dead cells and negative for live cells. See, FIG.6A through FIG. 6D.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforgoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

1. A compound according to the formula:

wherein X is independently —CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S; Y is independently carbon, nitrogen, —NR¹⁴, sulfur,oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S; Z is independently carbon, —CR¹², nitrogen, —NR¹⁴,sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C,CR¹²—CR¹³, NR¹⁴—NR¹⁵, or S—S; wherein at least one of X, Y and Z isother than carbon or C—C; R¹² is hydrogen, alkyl, substituted alkyl,alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde,carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; R¹³is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen,amino, substituted amino, aldehyde, carboxylic acid, ester, azido,nitro, nitroso, cyano, or thioether; R¹⁴ is hydrogen, alkyl, substitutedalkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino,aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, orthioether; R¹⁵ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid,ester, azido, nitro, nitroso, cyano, or thioether; R¹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹ comprisesa reactive group, carrier molecule or solid support; R² is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R² comprisesa reactive group, carrier molecule or solid support; R³ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R³ comprisesa reactive group, carrier molecule or solid support; R⁴ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁴ comprisesa reactive group, carrier molecule or solid support; R⁵ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁵ comprisesa reactive group, carrier molecule or solid support; R⁶ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁶ comprisesa reactive group, carrier molecule or solid support; R⁷ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁷ comprisesa reactive group, carrier molecule or solid support; R⁸ is hydrogen,alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁸ comprisesa reactive group, carrier molecule or solid support; R⁹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁹ comprisesa reactive group, carrier molecule or solid support; R¹⁰ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹⁰ comprisesa reactive group, carrier molecule or solid support; or a memberselected from R¹ in combination with R²; R² in combination with R³; R³in combination with R⁴; R⁴ in combination with R⁵; R⁶ in combinationwith R⁷ R⁷ in combination with R⁸; R⁸ in combination with R⁹; and R⁹ incombination with R¹⁰; together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl.
 2. The compound according to claim 1, wherein R⁸ is alkylsubstituted with —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂; wherein R′, R″, R′″ and R″″ each independently are selected fromthe group consisting of hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted alkyl, alkoxy, thioalkoxy and arylalkyl.
 3. The compoundaccording to claim 1, wherein R⁸ is —(CH₂)₃SO₃ ⁻.
 4. The compoundaccording to claim 1, wherein X is —CR¹², Y is nitrogen, and Z isoxygen.
 5. (canceled)
 6. The compound according to claim 1, wherein R⁴is —SO3⁻.
 7. The compound according to claim 1, wherein R³ is alkoxy. 8.The compound according to claim 1, wherein at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸ or R⁹ comprises a sulfo group.
 9. The compoundaccording to claim 1, wherein at least two of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸ or R⁹ comprises a sulfo group.
 10. The compound according toclaim 1, wherein at least three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ or R⁹comprises a sulfo group.
 11. The compound according to claim 1 whereinat least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ or R⁹ comprises areactive group, carrier molecule or solid support.
 12. The compoundaccording to claim 1, wherein R³ or R⁸ comprises a reactive group,carrier molecule or solid support. 13-19. (canceled)
 20. A method fordetecting an analye in a sample, wherein the method comprises a.combining a compound with a sample to form a combined sample, whereinthe compound has the formula

wherein X is independently —CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S; Y is independently carbon, nitrogen, —NR¹⁴, sulfur,oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S; Z is independently carbon, —CR¹², nitrogen, —NR¹⁴,sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C,CR¹²—CR¹³, NR¹⁴—NR¹⁵, or S—S; wherein at least one of X, Y and Z isother than carbon or C—C; R¹² is hydrogen, alkyl, substituted alkyl,alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde,carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; R¹³is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen,amino, substituted amino, aldehyde, carboxylic acid, ester, azido,nitro, nitroso, cyano, or thioether; R¹⁴ is hydrogen, alkyl, substitutedalkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino,aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, orthioether; R¹⁵ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid,ester, azido, nitro, nitroso, cyano, or thioether; R¹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹ comprisesa reactive group, carrier molecule or solid support; R² is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R² comprisesa reactive group, carrier molecule or solid support; R³ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R³ comprisesa reactive group, carrier molecule or solid support; R⁴ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁴ comprisesa reactive group, carrier molecule or solid support; R⁵ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁵ comprisesa reactive group, carrier molecule or solid support; R⁶ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁶ comprisesa reactive group, carrier molecule or solid support; R⁷ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁷ comprisesa reactive group, carrier molecule or solid support; R⁸ is hydrogen,alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁸ comprisesa reactive group, carrier molecule or solid support; R⁹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁹ comprisesa reactive group, carrier molecule or solid support; R¹⁰ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹⁰ comprisesa reactive group, carrier molecule or solid support; or a memberselected from R¹ in combination with R²; R² in combination with R³; R³in combination with R⁴; R⁴ in combination with R⁵; R⁶ in combinationwith R⁷ R⁷ in combination with R⁸; R⁸ in combination with R⁹; and R⁹ incombination with R¹⁰; together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; b. incubating the combined sample for a sufficient amount oftime for the compound to associate with the analyte in the sample toform an incubated sample; c. illuminating the incubated sample with anappropriate wavelength to form an illuminated sample; and d. detectingthe illuminated sample whereby the analyte in the sample is detected.21. The method according to claim 18, wherein the illumination and/ordetecting step comprises a flow cytometer. 22-23. (canceled)
 24. Amethod for evaluating the viability of cells in a sample, comprising a.combining a compound with a sample to form a combined sample, whereinthe compound comprises an amine reactive group and has the formula

wherein X is independently CR¹², nitrogen, —NR¹⁴, sulfur, oxygen,selenium, phosphorous, silicon, arsenic, amido, C—C, CR¹²—CR¹³,NR¹⁴—NR¹⁵, or S—S; Y is independently carbon, nitrogen, —NR¹⁴, sulfur,oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR12-CR13,NR14-NR15, or S—S; Z is independently carbon, —CR¹², nitrogen, —NR¹⁴,sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C,CR¹²—CR¹³, NR¹⁴—NR¹⁵, or S—S; wherein at least one of X, Y and Z isother than carbon or C—C; R¹² is hydrogen, alkyl, substituted alkyl,alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde,carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; R¹³is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen,amino, substituted amino, aldehyde, carboxylic acid, ester, azido,nitro, nitroso, cyano, or thioether; R¹⁴ is hydrogen, alkyl, substitutedalkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino,aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, orthioether; R¹⁵ is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy,sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid,ester, azido, nitro, nitroso, cyano, or thioether; R¹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹ comprisesa reactive group, carrier molecule or solid support; R² is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R² comprisesa reactive group, carrier molecule or solid support; R³ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R³ comprisesa reactive group, carrier molecule or solid support; R⁴ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁴ comprisesa reactive group, carrier molecule or solid support; R⁵ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁵ comprisesa reactive group, carrier molecule or solid support; R⁶ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁶ comprisesa reactive group, carrier molecule or solid support; R⁷ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁷ comprisesa reactive group, carrier molecule or solid support; R⁸ is hydrogen,alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁸ comprisesa reactive group, carrier molecule or solid support; R⁹ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R⁹ comprisesa reactive group, carrier molecule or solid support; R¹⁰ is hydrogen,alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino,substituted amino, aldehyde, carboxylic acid, ester, azido, nitro,nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring,substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R¹⁰ comprisesa reactive group, carrier molecule or solid support; or a memberselected from R¹ in combination with R²; R² in combination with R³; R³in combination with R⁴; R⁴ in combination with R⁵; R⁶ in combinationwith R⁷ R⁷ in combination with R⁸; R⁸ in combination with R⁹; and R⁹ incombination with R¹⁰; together with the atoms to which they are joined,form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-memberedheteroaryl; b. incubating the combined sample for a sufficient amount oftime for the compound to form a covalent bond with amine groups on acell membrane of live cells and to form covalent bonds with amine groupsinside dead cells in the sample to form an incubated sample; c.illuminating the incubated sample with an appropriate wavelength to forman illuminated sample; and d. detecting the illuminated sample wherebythe via of cells in a sample is evaluated.
 25. A kit for detecting ananalyte in a sample, wherein the kit comprises: a. a compound accordingto claim 1; and b. instructions for detecting the analyte.
 26. A kit forforming a dye conjugate, wherein the kit comprises: a compound accordingto claim 1, wherein the compound comprises a reactive group; and b.instructions for forming the dye conjugate with a carrier molecule orsolid support.